MAD4HATTER QC pipeline - Gambia_Indie
2023-09-18
Details of this dataset
Gambia_Indie-MH, 4 plates prepped between 2023_03 & 2023_04
Import data
Sample size, loci BEFORE cleanup
Explore data structure
## [1] "Sample size = 346"## [1] "Loci = 244"## [1] "1 to 130 alleles per locus per sample"Merging data
Combine multiple sequencing runs
Combine with parasite density
Combine with plate map
Plate map
Inspect all samples and controls in their spatial arrangement
This is a heatmap of average reads per locus for each sample
Trends in parasitemia
Combine with qPCR data (if available)
Check if libraries are balanced with parasite density
## `summarise()` has grouped output by 'sampleID', 'log_parasitemia'. You can
## override using the `.groups` argument.
## `summarise()` has grouped output by 'sampleID', 'log_parasitemia', 'plate'. You
## can override using the `.groups` argument.
Track reads through filtering
Estimates proportion of primer dimers
grey = input, colored = amplicons
## [1] "Percent dimers by sample prep date (in %, already multiplied by 100)"## # A tibble: 4 × 2
## prep_date dimer_percent
## <chr> <dbl>
## 1 2023_03_06 0.549
## 2 2023_03_28 0.356
## 3 2023_03_30 0.458
## 4 2023_04_05 0.825Negative controls
Check that the number of reads is low
Check if the reads found correspond to the pools you amplified (1A and 5)
## [1] "List of pool 1A/1B/2 loci with >100 reads (summed over all negative controls)"## # A tibble: 25 × 5
## locus reads pool Category Reason to include (if dr…¹
## <chr> <int> <chr> <chr> <chr>
## 1 Pf3D7_13_v3-2814583-2814832-2 344 2 <NA> HRP3 deletion
## 2 Pf3D7_11_v3-1902041-1902278-2 290 2 <NA> ch11
## 3 Pf3D7_13_v3-1726174-1726408-2 269 2 <NA> <NA>
## 4 Pf3D7_12_v3-2092654-2092884-2 249 2 <NA> 100
## 5 Pf3D7_13_v3-1725124-1725365-2 195 2 <NA> 613
## 6 Pf3D7_13_v3-1725887-1726123-2 176 2 <NA> <NA>
## 7 Pf3D7_13_v3-2770591-2770863-2 170 2 <NA> HRP3 deletion
## 8 Pf3D7_11_v3-1950431-1950680-2 169 2 <NA> ch11
## 9 Pf3D7_14_v3-294503-294768-2 160 2 <NA> cnv
## 10 Pf3D7_03_v3-221295-221532-2 159 2 <NA> C-terminal
## # ℹ 15 more rows
## # ℹ abbreviated name: ¹`Reason to include (if drug resistance: aminoacids)`Positive controls
Using a known parasite strain at a known density
Alleles should be known, and monoclonal
Presence of polyclonals suggest contamination => see plate map
<10 (arbitratry) reads or at <1% (arbitrary) of locus should be filtered out
## [1] "Mean of total locus = 214, Mean prop. of monoclonal loci = 0.85, Mean prop. of polyclonal loci = 0.15"## [1] "Pool 1A loci that are abset in positive controls:"## [1] "PKNH_12_v2-198869-199113-1AB" "PmUG01_12_v1-1397996-1398245-1AB"
## [3] "PocGH01_12_v1-1106456-1106697-1AB" "PvP01_12_v1-1184983-1185208-1AB"Look at total amplification
All pools combined
##
## 1A 1AB 1B 1B2 2
## 165 5 75 2 29
Species check
Controls should be Pfal, and not other species
NonPfal detection in samples suggests mixed infection (for downstream analysis)
These loci will be filtered out downstream (loci 1AB)
Look at individual pools
Pool 1A
## [1] "No reads in any samples for these loci, for 1A"## # A tibble: 0 × 3
## # ℹ 3 variables: locus <chr>, pool <chr>, n <dbl>## [1] "List of samples that do not have >75% loci with >100 reads, in pool 1A"## # A tibble: 172 × 7
## # Groups: sampleID [172]
## sampleID pool totreads n50 n100 neg_control norm
## <chr> <chr> <int> <int> <int> <lgl> <dbl>
## 1 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 235 0 0 FALSE 0
## 2 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 1989 4 0 FALSE 0
## 3 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 8057 61 18 FALSE 0.112
## 4 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 464 0 0 FALSE 0
## 5 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 989 0 0 FALSE 0
## 6 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 2786 7 0 FALSE 0
## 7 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 4984 28 6 FALSE 0.0375
## 8 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 7523 63 16 FALSE 0.1
## 9 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 9277 77 23 FALSE 0.144
## 10 Gambia_Indie-1B_MH_1_AE_2023_0… 1A 698 0 0 FALSE 0
## # ℹ 162 more rowsPool 5
##
## TRUE
## 47
## [1] "Loci without any reads, pool 5"## # A tibble: 0 × 16
## # ℹ 16 variables: locus-pool <chr>, Category <chr>, chr_malaria <chr>,
## # ampInsert_start <dbl>, ampInsert_end <dbl>, GeneID <chr>, Gene <chr>,
## # Reason to include (if drug resistance: aminoacids) <chr>,
## # Amino acid range (*: partial coverage of that aminoacid) <chr>,
## # pool5 <lgl>, amplicon_id <chr>, amplicon_start <dbl>, amplicon_end <dbl>,
## # fwd_primer <chr>, rev_primer <chr>, n <dbl>## [1] "List of samples that do not have >75% alleles with >100 reads, pool 5"## # A tibble: 154 × 7
## # Groups: sampleID [154]
## sampleID pool totreads n50 n100 neg_control norm
## <chr> <chr> <int> <int> <int> <lgl> <dbl>
## 1 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 86 0 0 FALSE 0
## 2 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 647 2 0 FALSE 0
## 3 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 3216 22 10 FALSE 0.227
## 4 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 138 0 0 FALSE 0
## 5 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 364 0 0 FALSE 0
## 6 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 862 2 0 FALSE 0
## 7 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 1550 14 3 FALSE 0.0682
## 8 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 2037 18 5 FALSE 0.114
## 9 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 3295 24 15 FALSE 0.341
## 10 Gambia_Indie-1B_MH_1_AE_2023_0… 1B 266 0 0 FALSE 0
## # ℹ 144 more rowsPool 2
Skip if not used
List samples that fail any of the QC checks
Reprep if <50% amplicons with >100 reads
Repool if >50% but <75% amplicons with >100 reads
## Joining with `by = join_by(sampleID)`Hypothetical filtering
Remove bad samples
Remove bad loci
Remove controls
## [1] "Sample size= 151"## [1] "Number of entries excluded = 0, Proportion excluded = 0"## [1] "Number of loci after cleanup = 242"## character(0)